The long-term objectives of this project are to elucidate the molecular structure of the mitochondrial citrate transport protein (CTP) at high resolution and to define the functions of specific amino acid residues in substrate recognition and translocation. This transporter catalyzes the exchange of tricarboxylates, dicarboxylates, and phosphoenolpyruvate across the inner mitochondrial membrane, land as such is essential to the energy metabolism of eukaryotic cells. Recently, we have: i) constructed a Cys-less CTP which displays functional properties that are nearly identical to the wild-type transporter; and ii) developed procedures enabling the purification of abundant quantities of the wild-type and Cys-less CTPs, as well as CTP mutants containing site-specifically engineered single Cys residues. From this foundation, we propose to initiate studies that will fundamentally advance our understanding of how this metabolically important transport protein functions at the molecular, chemical, and the atomic levels. Specifically, experiments will be conducted to: i) identify residues that comprise the substrate translocation pathway and/or are essential for CTP function using cysteine-substitution mutagenesis in combination with chemical modification of the resulting single Cys mutants; ii) determine the helix-helix and helix-loop proximities, tilt angles of helices, and ligand-induced conformational changes utilizing Cys-scanning mutagenesis in conjunction with site-directed thiol cross-linking and site- directed spin labeling; and iii) identify conditions enabling the growth of X-ray diffraction quality crystals and to subsequently determine the structure of the CTP. In combination, these studies will provide information essential to an understanding of the chemical and structural bases for Mitochondrial CTP function, and will provide the first high-resolution structure of a metabolite transport protein. The health relatedness of this project concerns the central role of the CTP in the bioenergetics of eukaryotic cells. Consequently, altered CTP function in disease (e.g., diabetes, cancer) is an important aspect of the aberrant intermediary metabolism that characterizes these pathologies. Thus, an elucidation of the chemical and structural bases for substrate translocation through the CTP is essential to an understanding of the role of the CTP in energy production in both normal physiological and pathological states.